Prognostic, diagnostic and clinicopathological roles of tsRNAs: a meta-analysis in breast cancer

Breast cancer (BC) is one of the most common malignancies in women and the leading cause of cancer-related death in women. The newly emerged non-coding RNAs tsRNAs (tRNA-derived small RNAs) play an important role in the occurrence and development of BC. The purpose of this study was to comprehensively evaluate the prognostic, diagnostic and clinicopathological roles of tsRNAs in BC. Through literature screening, a total of 13 BC-related tsRNA studies were included in this meta-analysis, all of which passed quality assessment. Prognostic studies showed upregulated tsRNAs to be associated with poor survival outcomes (HR = 1.64, 95%CI 1.51–1.77) and downregulated tsRNAs to be associated with better outcomes (HR = 0.58, 95%CI 0.50–0.68). Results of diagnostic studies showed a combined sensitivity of 72% (95%CI 68–76%) and combined specificity of 64% (95%CI 61–67%); the AUC was 0.72 (95%CI 0.68–0.75) and the DOR 4.62 (95%CI 3.76–5.68). Finally, correlation analysis of clinicopathological features showed that downregulation of tsRNAs correlated significantly with age, TNM stage and lymphatic metastasis. Sensitivity analysis and publication bias showed no significant difference. In conclusion, BC-associated tsRNAs are closely related to the prognosis and clinicopathological features of patients with this disease and can be used to assist in early diagnosis of BC. Therefore, tsRNAs are potential targets for the diagnosis and treatment of BC. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-023-01617-2.


Introduction
According to the latest cancer statistics data, breast cancer (BC) is the most common malignant tumor in women, accounting for 31% of the total number of female cancers, and the incidence has gradually increased in recent years [1,2].With continuous improvement in diagnosis and treatment of BC, early-stage patients have good prognosis, with an overall cure rate of 90% [3].
However, the 5-year survival rate is significantly reduced for patients with advanced stage, poor tissue typing or resistance to combined therapy [4].Therefore, clarifying the specific molecular mechanism of the occurrence and development of BC to assist in early diagnosis, as well as to find new and more accurate targeted molecules, is crucial for improving the overall survival rate of BC patients.
In recent years, positive roles of a variety of non-coding RNAs (ncRNAs), including micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and tRNA-derived small RNAs (tsRNAs), in cancer have received much attention and been widely reported; some have been included in clinical application, fully demonstrating the huge potential of ncRNAs in tumor diagnosis and treatment [5][6][7].TsRNAs are the products of tRNA or pre-tRNA cleavage during maturation.TsRNAs can be divided according to different fracture sites into tRNArelated fragments (tRFs) and tRNA halves (tiRNAs), which complement each other in terms of formation pathway, cell localization and function [8,9].Although tsRNAs were initially thought to be produced by random degradation and have no special function, an increasing number of studies have shown that tsRNAs are not only widely expressed in tumors but also play important roles in the occurrence and development of tumors [10].In addition, tsRNAs are present in a large number of human body fluids, such as saliva, urine, and semen, providing a new target for early tumor diagnosis and precise treatment [11].
Studies have shown that tsRNAs are expressed specifically in BC and widely exist in the serum of BC patients.TsRNAs differentially expressed in different subtypes of BC, including triple-negative breast cancer, have been mined through RNA sequencing and verified with clinical samples, suggesting the significance of tsRNAs in identifying tumor properties and tumor tissue types [12][13][14].In terms of mechanism research, tsRNAs are significantly enriched in hormone-dependent BC and are not only directly involved in regulating the occurrence and development of BC but also closely related to drug resistance and tumor recurrence in BC [15][16][17].Overall, tsRNAs are good detection markers and potential therapeutic targets for BC patients.Therefore, we summarized existing studies on BC and tsRNAs and used meta-analysis to explore the roles and values of tsRNAs in the prognosis, diagnosis and clinicopathological features of BC patients to provide evidence for future applications of tsRNAs.

Publication search
To collect relevant research literature for meta-analysis, the Boolean logic method was used to search PubMed, Web of Science, Cochrane Library, Embase and other databases as of March 1, 2023.Specific search terms included "breast cancer", "breast carcinoma", "tsRNA", "tRNA-derived small RNA", "tRF", "tRNA-derived fragment", "tiRNA" and "tRNA-derived stress-induced RNA".

Inclusion and exclusion criteria
Inclusion criteria: (1) the content included the relationship between tsRNAs and BC; (2) all cases passed the gold standard, that is, the histopathological diagnosis was clear; and (3) data related to prognosis, diagnosis or clinicopathological features could be extracted directly or indirectly.Exclusion criteria: (1) the content was not related to tsRNAs or BC; (2) reviews, meta-analyses, letters, case reports, conference abstracts; (3) non-English and nonhuman studies; and (4) failure to report or extract important indicators and data.Two researchers independently read the title and abstract of the retrieved literature for preliminary screening; in cases in which the abstract results were not clear, the full text was read to determine inclusion eligibility.If the screening results were inconsistent, the third researcher decided.

Data extract
The general data extracted from each article included the following: title, first author, tsRNAs, year, country, expression, cut-off, sample size, data source, detected sample, among others.Data related to prognosis included follow-up time, survival outcome (progression-free survival (PFS), recurrence-free survival (RFS), disease-free survival (DFS), overall survival (OS)), survival analysis, hazard ratio (HR), and 95% confidence interval (95%CI), among others.Data related to diagnosis included case, control, area under the receiver operating characteristic curve (AUC), sensitivity (SEN), specificity (SPE), true positive (TP), false positive (FP), true negative (TN), false negative (FN), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and cancer type, among others.Data related to clinicopathologic features included age, TNM stage, and lymphatic metastasis, among others.All records were approved by two independent researchers.

Quality assessment
As the quality of studies may influence the results of meta-analysis, each included study was evaluated using the Newcastle-Ottawa score (NOS) tables in the Cochrane manual and Quality Assessment for Studies of Diagnostic Accuracy 2 (QUADAS 2).After all evaluations, RevMan 5.3 software was used to output the evaluation results.NOS scores ≥ 7 or QUADAS 2 scores ≥ 4 are generally considered to be high quality [18,19].

Data synthesis and analysis
Statistical analysis of the data was performed with Stata software (version 15.1).HRs, odds ratios (ORs), and 95%CIs were used to evaluate the effect of tsRNAs on prognosis and clinicopathologic features in BC patients.To evaluate the diagnostic value of tsRNAs, summary receiver operating characteristic (SROC) curves were drawn, and AUCs were calculated, as were Fagan nomograms and scatter plots.For studies in which Kaplan-Meier (KM) and ROC curve data could not be extracted directly, we used Engage Digitizer and GetData Graph Digitizer and calculated HR and the corresponding 95%CI based on the method of Tierney et al. [20][21][22].The Cochran-Q test and I 2 statistics were used to assess heterogeneity.When the heterogeneity was small (P > 0.10 or I 2 < 50%), a meta-analysis was conducted using the fixed effects model.Otherwise, random-effects models were employed to combine effect sizes, and further subgroup analysis and meta-regression (MetaDiSc software) were applied to explore possible sources of interstudy heterogeneity.Sensitivity analysis was carried out by excluding the included studies one by one.Finally, Begg's funnel plot, Deeks' funnel plot and Egger's test were used to evaluate publication bias, and P > 0.05 was considered to indicate no publication bias.All statistical tests were bilateral, and P < 0.05 was considered statistically significant.
NOS and QUADAS 2 scoring methods were used to systematically assess the quality of all included prognostic and diagnostic studies.The results showed that the quality of all the included studies met the standards.Risk of bias graphs and summaries are shown in Additional file 1: Figures S1 and Additional file 2: Figure S2.

Prognostic roles of tsRNAs in BC
There were seven studies, all of which were conducted in China except for one in the USA and published between 2018 and 2022.The sample type used for detection was mainly tissue, with a total of 5257 patients.Clinical Finally, 10 high expression tsRNAs and 6 low expression tsRNAs related to BC were identified (Table 1).
The fixed effect model was used to evaluate the correlation between tsRNAs and prognosis of BC patients according to the level of heterogeneity.According to forest map results, high expression of tsRNAs was associated with poor clinical outcomes (HR = 1.64, 95%CI 1.51-1.77).Conversely, low expression of tsRNAs was associated with better clinical outcomes (HR = 0.58, 95%CI 0.50-0.68).These results indicate that tsRNAs may be important prognostic factors for BC (Fig. 2).In addition, subgroup analysis of differentially expressed tsRNAs was performed, and the results showed that parameters such as country, cut-off, sample size, data source, detected sample, follow-up time, survival outcome, survival analysis, and HR obtained were associated with BC prognosis (Additional file 3: Figure S3 and Additional file 4: Figure S4).

Diagnostic roles of tsRNAs in BC
In Table 2, the SEN, SEP, LPR, NPR and DOR of BC diagnosis are summarized, and the results are shown in the form of a forest map.The combined SEN (Fig. 3A) was 72% (95%CI 68-76%), and the combined SEP (Fig. 3B) was 64% (95%CI 61-67%).The combined PLR (Fig. 3C) was 2.00 (95%CI 1.83-2.20)and the combined NLR (Fig. 3D) 0.43 (95%CI 0.38-0.50).The DOR (Fig. 3E) was 4.62 (95%CI 3.76-5.68).Subsequently, we plotted the SROC curve (Fig. 3G), and the AUC was 0.72 (95%CI 0.68-0.75).These results suggest that tsRNAs have good efficacy in diagnosis of BC and have potential to be used as diagnostic markers.Moreover, bivariable box diagram results (Fig. 4A) showed that most studies were distributed within the 95%CI.To analyze sources of heterogeneity among the studies, we evaluated threshold effects.The results showed that the SROC curve graph (Fig. 3F) did not show a typical "shoulder-arm" distribution, considering that there was no threshold effect.Covariables country, expression, case, and type were used for subgroup analysis (Table 3) and meta-regression analysis (Table 4) to evaluate the nonthreshold effects.The I 2 of the DOR for country (China vs. Japan) was 53.5% vs. 0.0%, and the I 2 of the DOR for expression (Down vs. Up vs. NA) was 62.9% vs. 40.7% vs. 0.0%, partially reducing heterogeneity.However, in univariate multiple regression analysis, the covariables were all P > 0.05, no significant correlation was found with DOR, and heterogeneity due to experimental design was not considered.
To further evaluate the ability of tsRNAs as diagnostic markers in patients with BC, Fagan's nomogram (Fig. 4C) and Scatter plots of PLR and NLR (Fig. 4D) were developed.When the prior probability is 20%, the probability of BC in positive tsRNAs test increases to 33%, and the probability of BC in negative tsRNAs test decreases to 10%.All the above results indicate that tsRNAs have high diagnostic capability of BC and are a good diagnostic test.

Sensitivity analysis and publication bias
To assess the impact of each study on the results of the meta-analysis, sensitivity analysis was performed on the included literature.The results showed no significant change in the combined total HRs or ORs after the removal of each study, suggesting little difference among studies and relatively stable results (Additional file 4: Figure S4 and Additional file 5: Figure S5).
Begg's funnel plots and the Egger test were used to assess the existence of publication bias.The funnel plots of prognostic correlation studies were roughly symmetric (Additional file 1: Figure S4).However, one of the lymphatic metastasis indexes in the clinicopathological feature correlation analysis fell outside the funnel plot, suggesting publication bias in the included studies, which may be related to the small amount of data (Additional file 5: Figure S5).Finally, Deeks' funnel plots were applied to analyze publication bias in diagnostic correlation studies.The results showed a basically symmetrical funnel plot (P = 0.59), suggesting no significant publication bias (Fig. 4B).

Discussion
BC is the malignant tumor with the highest incidence in the female population at present [35,36].Its causes are still unclear, and some types lack effective intervention targets, resulting in a high fatality rate.Traditional BC detection methods, such as CEA, CA153, B-ultrasound, and molybdenum targeted therapy, among others, are relatively less traumatic than other methods but have low sensitivity and specificity for early diagnosis, and pathology, the gold standard of diagnosis, is difficult to popularize in the population without disease [37][38][39][40].Therefore, exploration of more effective markers for early diagnosis Table 1 Characteristics of all prognostic studies included in the meta-analysis  As emerging ncRNAs, abnormal expression of tsRNAs has been confirmed in a variety of diseases, including cancers, and plays an important biological role and function [41,42].As tRNA fragments, tsRNAs are characterized by low molecular weight and stable expression.In addition, tsRNAs are widely expressed and specific in human tissues and body fluids.These characteristics all render tsRNAs important measures in cancer diagnosis and prognosis prediction [43,44].Since Hani et al. discovered that tRFs derived from tRNA-Glu, tRNA-Asp, tRNA-Gly, and tRNA-Tyr competitively bind to the endogenous oncogene YBX1 in BC cells, inhibiting cell growth by interfering with oncogene transcription stability, the role and mechanism of tsRNAs in BC occurrence and development have been extensively explored [15].Wang et al. conducted tsRNA expression sequencing on plasma samples from 8 BC patients and 4 healthy women, and found that the expression level of tRF-Glu-CTC-003 in BC patients' plasma was lower compared to that in healthy women, and the expression level of tRF-Glu-CTC-003 in TNBC patients' plasma was lower than in other subtypes [31].These differential results suggest that tsRNA may serve as a potential biomarker for BC.Detecting tsRNA levels in plasma can aid in early BC diagnosis and patient prognosis evaluation.Mechanistically, tsRNAs primarily regulate protein expression at different stages by binding to other molecules, thereby affecting protein biosynthesis through transcription or post-transcriptional processes, and playing a regulatory role in BC.Maurizio et al. found significantly lower expression levels of tRF3E derived from mature tRNA-Glu in BC tissues compared to normal tissues, which could inhibit BC progression by binding to the RNA-binding protein NCL [45].Zhu et al. identified high expression of tRF-Lys-CTT-010 in TNBC, demonstrating its ability to promote cell proliferation and migration, participate in metabolic pathways, To our knowledge, this is the first meta-analysis exploring the value of tsRNAs in BC, with a view to providing evidence-based medical evidence for future clinical applications of tsRNAs.According to our inclusion and exclusion criteria, 13 studies on the correlation between tsRNAs and BC were identified as eligible for meta-analysis, and no studies that did not meet the criteria were found after quality assessment.In the study on the correlation between tsRNAs and BC patient prognosis, we included 7 studies involving a total of 5257 patients.The results showed that tsRNA expression correlated positively with prognostic indicators (PFS, RFS, DFS, OS), which was consistent with the study on the mechanism of differentially expressed tsRNAs playing a role in promoting or suppressing BC.It should be noted that the cut-off method for classifying tsRNA expression was not unified among the included studies, and there is still a lack of quantitative analysis standards for tsRNAs, posing challenges for practical clinical application of tsRNAs in the future.In addition, the vast majority of studies focused on Asia, and more solid research is needed to confirm whether our results are biased by race and/or region.Wang et al. found that the AUC of the combination of six tsRNAs in the plasma of early-stage BC patients was 0.844, superior to any single identified tsRNA [32].In addition, Zhang et al. reported that when three tsRNAs were combined with the traditional tumor markers CEA, CA125 and CA153, the AUC increased to 0.801 [33].These results suggest that tsRNAs have reliable potential for BC diagnosis.Therefore, we systematically reviewed studies on the diagnostic value of tsRNAs in BC, aiming to identify biomarkers that can be used for diagnosis.Regarding clinicopathological studies, 3 studies were included in total.The results showed that low expression of tsRNAs correlated significantly with age, TNM stage and lymphatic metastasis, suggesting a role in inhibiting BC progression.In addition, we found a certain publication bias in the included studies, which affected the accuracy of the results obtained; the reason may be that there were few studies and that some of them had small sample sizes.For example, in the study of Wang et al. ts-32-Q99P9P9NH57SJ and ts-17-79MP9PP showed low expression in BC, though the trend in lymphatic metastasis was the opposite, which may also be related to the small number of samples (n = 16) [28].In conclusion, we believe that tsRNAs may become a new target in BC treatment and provide new ideas for BC treatment.However, more literature needs to be examined to obtain reliable data support.At present, research on tsRNAs is still in the initial stage, and the specific molecular mechanism of the role of a large number of differentially expressed tsRNAs in tumors, especially BC, has not been clarified.Nevertheless, we hope that our meta-analysis will be helpful for future clinical applications of tsRNAs.Of course, there are several limitations of this study that should not be ignored.First, as the patients included were mostly from China, the conclusion may not be applicable to different regions or populations.Second, some data and their 95%CIs were obtained indirectly through software, which may lead to deviation from the real data, resulting in certain publication bias.In addition, there are few related studies on different subtypes, especially triple-negative breast cancer; thus, the positive role of tsRNAs in the early diagnosis and prognosis of these patients should be explored.Finally, comprehensive treatment tolerance is one of the main factors affecting the survival of BC patients at present, but there are few studies focusing on such factors.Future studies with more and larger samples should be designed and performed to guide individualized clinical therapy (Additional file 6: Figure S6).

Conclusion
In summary, our study found that tsRNAs have important value in diagnosis and prognosis assessment of BC and correlate significantly with some clinicopathological features, suggesting that tsRNAs can be used as an effective diagnostic and treatment marker for BC patients.However, due to the limited sample size, the results may be unstable.Therefore, more high-quality research data are needed to support or update our conclusions in the future.

Fig. 1
Fig. 1 Workflow of the study and result of document screening

35 Fig. 2
Fig. 2 Forest plots of upregulated (A) and downregulated (B) tsRNAs for survival outcome in breast cancer

Table 2
Characteristics of all diagnostic studies included in the meta-analysis a AUC: the area under the receiver operating characteristic curve; b Sen: sensitivity; c Spe: specificity; d TP: true positive; e FP: false positive; f TN: true negative; g FN: false negative; h PLR: positive likelihood ratio; i NLR: negative likelihood ratio; j DOR: diagnostic odds ratio; k Ref: reference; l BC: breast cancer; m NTNBC: non-triple negative breast cancer; n EBC: early-stage breast cancerNo Study

Year Country Expre ssion Sample size Variables Cancer type Ref k Case Control AUC a Sen b
and precise treatment is crucial for improving the poor prognosis of BC patients.

Table 3
Assessment of diagnostic accuracy and heterogeneity in subgroup analysisa BC: breast cancer; b EBC: early-stage breast cancer; c NTNBC: non-triple negative breast cancer

Table 4
Results of univariate meta-regression analysis of diagnostic odds ratio

Table 5
Meta-analyses of correlation between downregulated tsRNAs and clinicopathological features of breast cancer

Downregulated tsRNAs No. of studies No. of patients Odds ratio (95%CI) p-value Heterogeneity I 2 (%)
Fig. 5 Forest plots of age (A), TNM stage (B) and lymphatic metastasis (C) of tsRNAs in breast cancer